scholarly journals The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Manoel L. Costa ◽  
Arnon D. Jurberg ◽  
Claudia Mermelstein
Keyword(s):  
Animal Models ◽  
Cell Lines ◽  
Muscle Cell ◽  
Fruit Fly ◽  
Embryonic Chick ◽  
Skeletal Myogenesis ◽  
And Migration ◽  
High Level

The mechanisms involved in the development of skeletal muscle fibers have been studied in the last 70 years and yet many aspects of this process are still not completely understood. A myriad of in vivo and in vitro invertebrate and vertebrate animal models has been used for dissecting the molecular and cellular events involved in muscle formation. Among the most used animal models for the study of myogenesis are the rodents rat and mouse, the fruit fly Drosophila, and the birds chicken and quail. Here, we describe the robustness and advantages of the chick primary muscle culture model for the study of skeletal myogenesis. In the myoblast culture obtained from embryonic chick pectoralis muscle it is possible to analyze all the steps involved in skeletal myogenesis, such as myoblast proliferation, withdrawal from cell cycle, cell elongation and migration, myoblast alignment and fusion, the assembly of striated myofibrils, and the formation of multinucleated myotubes. The fact that in vitro chick myotubes can harbor hundreds of nuclei, whereas myotubes from cell lines have only a dozen nuclei demonstrates the high level of differentiation of the autonomous chick myogenic program. This striking differentiation is independent of serum withdrawal, which points to the power of the model. We also review the major pro-myogenic and anti-myogenic molecules and signaling pathways involved in chick myogenesis, in addition to providing a detailed protocol for the preparation of embryonic chick myogenic cultures. Moreover, we performed a bibliometric analysis of the articles that used this model to evaluate which were the main explored topics of interest and their contributors. We expect that by describing the major findings, and their advantages, of the studies using the embryonic chick myogenic model we will foster new studies on the molecular and cellular process involved in muscle proliferation and differentiation that are more similar to the actual in vivo condition than the muscle cell lines.

scholarly journals Progression and Differentiation of Alveolar Rhabdomyosarcoma Is Regulated by PAX7 Transcription Factor—Significance of Tumor Subclones

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1870
Author(s):  
Klaudia Skrzypek ◽  
Grażyna Adamek ◽  
Marta Kot ◽  
Bogna Badyra ◽  
Marcin Majka
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Migration Activity ◽  
Id Proteins ◽  
Rh30 Cell ◽  
Str Profile ◽  
And Migration

Rhabdomyosarcoma (RMS), is the most frequent soft tissue tumor in children that originates from disturbances in differentiation process. Mechanisms leading to the development of RMS are still poorly understood. Therefore, by analysis of two RMS RH30 cell line subclones, one subclone PAX7 negative, while the second one PAX7 positive, and comparison with other RMS cell lines we aimed at identifying new mechanisms crucial for RMS progression. RH30 subclones were characterized by the same STR profile, but different morphology, rate of proliferation, migration activity and chemotactic abilities in vitro, as well as differences in tumor morphology and growth in vivo. Our analysis indicated a different level of expression of adhesion molecules (e.g., from VLA and ICAM families), myogenic microRNAs, such as miR-206 and transcription factors, such as MYOD, MYOG, SIX1, and ID. Silencing of PAX7 transcription factor with siRNA confirmed the crucial role of PAX7 transcription factor in proliferation, differentiation and migration of RMS cells. To conclude, our results suggest that tumor cell lines with the same STR profile can produce subclones that differ in many features and indicate crucial roles of PAX7 and ID proteins in the development of RMS.

scholarly journals EXTH-64. SMALL GTPASES IN MENINGIOMAS: PROLIFERATION, MIGRATION, SURVIVAL, POTENTIAL TREATMENT AND INTERACTIONS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii101-ii101
Author(s):  
Christoph Kesseler ◽  
Julian Kahr ◽  
Natalie Waldt ◽  
Nele Stroscher ◽  
Josephine Liebig ◽  
...  
Keyword(s):  
Overall Survival ◽  
Cell Lines ◽  
Small Gtpases ◽  
Potential Treatment ◽  
Rock Inhibitor ◽  
Meningioma Cell ◽  
And Migration ◽  
Proliferation And Migration

Abstract PURPOSE To evaluate the role of the small GTPases RhoA, Rac1 and Cdc42 in meningiomas as therapeutic targets and their interactions in meningiomas. EXPERIMENTAL DESIGN We analyzed expression of GTPases in human meningioma samples and meningioma cell lines of various WHO grades. Malignant IOMM-Lee meningioma cells were used to generate shRNA mediated knockdowns of GTPases RhoA, Rac1 or Cdc42 and to study knockdown effects on proliferation and migration, as well as analysis of cell morphology by confocal microscopy. The same tests were used to investigate effects of the two inhibitors Fasudil and EHT-1864 of malignant IOMM-Lee, KT21 and benign Ben-Men cells and the effects of these drugs on IOMM-Lee knockdown cells. The effects of GTPase knockdowns and Fasudil treatment were studied in terms of overall survival by intracranial xenografts of mice. Potential interactions of GTPases regarding NF2, mTOR and FAK-Paxillin were examined. RESULTS Small GTPases were upregulated in meningiomas of higher tumor grades. Reduced proliferation and migration could be achieved by GTPase knockdown in IOMM-Lee cells. Additionally, the ROCK-inhibitor Fasudil and Rac1-inhibitor EHT-1864 reduced proliferation in different meningioma cell lines and reduced proliferation and migration independent of GTPase knockdowns/status. Moreover, overall survival in vivo could also be increased by knockdowns of RhoA and Rac1 as well as Fasudil treatment. GTPase expression was affected dependent on the NF2 status but effects were not very distinct, indicating that NF2 is not strongly involved in GTPase regulation in meningiomas. In terms of mTOR and FAK-Paxillin signaling, each GTPase changes those pathways in a different manner. CONCLUSION Small GTPases are important effectors in meningioma proliferation and migration in vitro as well as survival in vivo and their inhibition should be considered as potential treatment option.

scholarly journals BSCI-12. Inhibition of melanoma brain metastasis by targeting miR-146a

2021 ◽  
Vol 3 (Supplement_3) ◽  
pp. iii3-iii3
Author(s):  
Jiwei Wang ◽  
Emma Rigg ◽  
Taral R Lunavat ◽  
Wenjing Zhou ◽  
Zichao Feng ◽  
...  
Keyword(s):  
Brain Metastasis ◽  
Tumor Cells ◽  
Cell Lines ◽  
Western Blots ◽  
Cell Growth And Migration ◽  
Human Astrocytes ◽  
And Migration ◽  
The Brain

Abstract Background Melanoma has the highest propensity of any cancer to metastasize to the brain, with late-stage patients developing brain metastasis (MBM) in 40% of cases. Survival of patients with MBM is around 8 months with current therapies, illustrating the need for new treatments. MBM development is likely caused by molecular interactions between tumor cells and the brain, constituting the brain metastatic niche. miRNAs delivered by exosomes released by the primary tumor cells may play a role in niche establishment, yet the mechanisms are poorly understood. Here, the aim was to identify miRNAs released by exosomes from melanomas, which may be important in niche establishment and MBM progression. Materials and Methods miRNAs from exosomes collected from human astrocytes, melanocytes, and MBM cell lines were profiled to determine differential expression. Functional in vitro validation was performed by cell growth and migration assays, cytokine arrays, qPCR and Western blots. Functional in vivo studies were performed after miR knockdown in MBM cell lines. An in silico docking study was performed to determine drugs that potentially inhibit transcription of miR-146a to impede MBM development. Results miR-146a was the most upregulated miRNA in exosomes from MBM cells and was highly expressed in human and animal MBM samples. miR-146a mimics activated human astrocytes, shown by increased proliferation and migration, elevated expression of GFAP in vitro and in mouse brain tumor samples, and increased cytokine production. In animal studies, knockdown of miR-146a in MBM cells injected intracardially into mice reduced BM burden and increased animal survival. Based on the docking studies, deserpidine was found to be an effective inhibitor of MBM growth in vitro and in vivo. Conclusions MiR-146a may play an important role in MBM development, and deserpidine is a promising candidate for clinical use.

scholarly journals Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 930-936 ◽  
Author(s):  
I Max-Audit ◽  
U Testa ◽  
D Kechemir ◽  
M Titeux ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Cell Lines ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Low Concentrations ◽  
Erythroleukemia Cell ◽  
Erythroleukemic Cell ◽  
High Level

To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R- PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.

P16.08 Inhibition of melanoma brain metastasis by targeting miR-146a

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii57-ii57
Author(s):  
J Wang ◽  
E K Rigg ◽  
T R Lunavat ◽  
W Zhou ◽  
Z Feng ◽  
...  
Keyword(s):  
Brain Metastasis ◽  
Tumor Cells ◽  
Cell Lines ◽  
Western Blots ◽  
Cell Growth And Migration ◽  
Human Astrocytes ◽  
And Migration ◽  
The Brain

Abstract BACKGROUND Melanoma has the highest propensity of any cancer to metastasize to the brain, with late-stage patients developing brain metastasis (MBM) in 40% of cases. Survival of patients with MBM is around 8 months with current therapies, illustrating the need for new treatments. MBM development is likely caused by molecular interactions between tumor cells and the brain, constituting the brain metastatic niche. miRNAs delivered by exosomes released from the primary tumor cells may play a role in niche establishment, yet the mechanisms are poorly understood. Here, the aim was to identify miRNAs released by exosomes from melanomas, which may be important in niche establishment and MBM progression. MATERIAL AND METHODS miRNAs in exosomes collected from human astrocytes, melanocytes, and MBM cell lines were profiled to determine differential expression. Functional in vitro validation was performed by cell growth and migration assays, cytokine arrays, qPCR and Western blots. Functional in vivo studies were performed after miR knockdown in MBM cell lines. An in silico docking study was performed to determine drugs that potentially inhibit transcription of miR-146a to impede MBM development. RESULTS miR-146a was the most upregulated miRNA in exosomes from MBM cells and was highly expressed in human and animal MBM samples. miR-146a mimics activated human astrocytes, shown by increased proliferation and migration, elevated expression of GFAP in vitro and in mouse brain tumor samples, and increased cytokine production. In animal studies, knockdown of miR-146 in MBM cells injected intracardially into mice reduced BM burden and increased animal survival. Based on the docking studies, deserpidine was found to be an effective inhibitor of MBM growth in vitro and in vivo. CONCLUSION miR-146a may play an important role in MBM development, and deserpidine is a promising candidate for clinical use.

Glutathione S-transferases in tracheobronchial epithelium

1995 ◽  
Vol 269 (4) ◽  
pp. L473-L481
Author(s):  
P. M. Reddy ◽  
C. P. Tu ◽  
R. Wu
Keyword(s):  
Cell Differentiation ◽  
Environmental Factors ◽  
Enzymatic Activity ◽  
Cell Lines ◽  
Mucous Cell ◽  
Collagen Gel ◽  
Bronchial Epithelium ◽  
High Level

The purpose of this study is to characterize glutathione S-transferase (GST) gene expression in airway epithelium both in vivo and in vitro. Immunohistochemical staining of nonhuman primate lungs of well-controlled healthy animals reveals the presence of alpha- and pi-class GST isoenzymes in ciliated bronchial epithelium. The stain of mu-GST antibody is either very low or absent in some of these monkey lungs. We observed that primary tracheobronchial epithelial (TBE) cells isolated from human and monkey pulmonary tissues maintain a relatively high level of GST enzymatic activity in culture, compared with various immortalized human TBE cell lines and other nonpulmonary cell lines. Northern blot analysis demonstrated the presence of mu-, pi-, and microsomal-GST messages but not the alpha-class message in cultures of primary TBE cells as well as in various human TBE cell lines. The expression of mu- and pi-class GST genes can be further regulated in culture by various environmental factors; however, most of these regulating factors are associated with TBE cell differentiation in culture. For instance, vitamin A treatment, which was shown to enhance mucous cell differentiation in vitro, stimulated the message levels of mu- and pi-class GST. Furthermore, plating cells on collagen gel substrata, which also enhanced mucous cell differentiation in culture, instead of plastic culture surface, enhanced total GST enzymatic activity by eightfold, and this enhancement is related to an increase in the expression of the pi-class GST gene. These results demonstrated that GST genes are differentially expressed and regulated by various environmental factors in primary TBE cells and various cell lines, and the regulation is correlated to the mucous cell differentiation in culture.

scholarly journals Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 930-936 ◽  
Author(s):  
I Max-Audit ◽  
U Testa ◽  
D Kechemir ◽  
M Titeux ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Cell Lines ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Low Concentrations ◽  
Erythroleukemia Cell ◽  
Erythroleukemic Cell ◽  
High Level

Abstract To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R- PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.

ANKHD1 Silencing Reduces In Vitro Clonogenicity and In Vivo Tumorogenicity Of Multiple Myeloma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3168-3168
Author(s):  
Anamika Dhyani ◽  
João Agostinho Machado-Neto ◽  
Patricia Favaro ◽  
Sara Teresinha Olalla Saad
Keyword(s):  
Cell Cycle ◽  
Gene Therapy ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Tumor Size ◽  
Control Group ◽  
Mice Model ◽  
And Migration

Abstract Introduction ANKHD1 is a multiple ankyrin repeats containing protein, highly expressed in cancers, such as acute leukemia. Earlier studies showed that ANKHD1 is highly expressed and plays important role in proliferation and cell cycle progression of multiple myeloma (MM) cells. It was also observed that ANKHD1 downregulation modulates cell cycle gene expression and upregulates p21 irresepective of TP53 mutational status of MM cell lines. Objective The present study aimed to study the effect ofANKHD1 silencing on MM growth both in vitro (clonogenicity, migration) and in vivo (xenograft tumor mice model). The purpose was to investigate the feasibility of ANKHD1 gene therapy for MM. Methods In the present study, ANKHD1 expression was silenced using short hairpin RNA (shRNA)-lentiviral delivery vector in MM cell lines (U266 and MM1S). For control MM cells were tranduced by lentiviral shRNA against LacZ. Downregulation of ANKHD1 expression was confirmed by qPCR and Western blot. Colony formation capacity and migration of control and ANKHD1 silenced MM cells was determined by methylcellulose and transwell migration assays, respectively. For in vivo MM growth, NOD-SCID mice were divided in two groups injected with control and ANKHD1 silenced cells, separately. Mice were observed daily for tumor growth. Once the tumor size reached 1 mm3, mice in both groups were sacrificed and tumor was excised to measure tumor volume and weight. Results Corroborating the results obtained in our earlier studies, in the present study also inhibition of ANKHD1 expression suppressed growth of MM cells in vitro. MM cell lines tranduced with ANKHD1 shRNA showed significantly low number of colonies ten days after plating in methylcellulose medium as compared to control (p<0.05). Similarly, in transwell migration assay, cell lines transduced with ANKHD1 showed significantly less migration as in response to 10% FBS at lower chamber as compared to control group (p<0.05) in both the cell lines analyzed. Further in xenograft MM mice model, the growth of tumor was visibly suppressed in mice injected with ANKHD1 silenced cells compared to control group. There was significant difference in tumor size (volume) between these 2 groups (P< 0.006). The tumor weight of the inhibition group was 0.71 ±0.2 g, significantly lighter than those of the control group (1.211 ± 0.5 g, P =0.02) Conclusion Our data indicates ANKHD1 downregulation significantly inhibits colony-forming ability and migration of both glucocorticoid resistant (U266) and sensitive (MM1S) MM cells. Further, gene silencing of ANKHD1 also resulted in reduced in vivo tumor growth in NOD/SCID mice. Collectively, the result obtained indicates that ANKHD1 may be a target for gene therapy in MM. Disclosures: No relevant conflicts of interest to declare.

Silencing The Sialyltransferase Gene ST3GAL6 Inhibits Adhesion and Migration Of Myeloma Cells In Vitro and Reduces The Homing and Proliferation Of Tumor Cells In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 275-275
Author(s):  
Siobhan Glavey ◽  
Salomon Manier ◽  
Antonio Sacco ◽  
Michaela R Reagan ◽  
Yuji Mishima ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Board ◽  
Myeloma Cells ◽  
Sialyl Lewis ◽  
And Migration ◽  
Rpmi 8226

Abstract Background Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The adhesion and trafficking of multiple myeloma (MM) cells is strongly influenced by glycosylation and multiple myeloma cells express a variety of adhesion molecules, including selectin ligands and integrins, which are typically dependent on glycosylation for their function. We have previously reported that the sialyltransferase ST3GAL6 is up-regulated in plasma cells from MM patients and that increased expression is associated with inferior overall survival (OS) in MM gene expression profiling (GEP) datasets. The functional significance of increased sialylation of MM cells has not previously been reported. Methods MM cell lines MM1s and RPMI-8226 were confirmed to have high expression levels of ST3GAL6 at the gene and protein level compared to healthy controls. Knockdown of ST3GAL6 was confirmed in MM cell lines RPMI-8226 and MM1s using lentiviral shRNAs targeting different regions in the ST3GAL6 mRNA. Specific ST3GAL6 knockdown was confirmed by reduced ST3GAL6 mRNA and protein expression in comparison to a scrambled control. In a calcein-AM fluorescence based adhesion assay we next evaluated the effects of ST3GAL6 knockdown on MM-cell adhesion to bone marrow stromal cells (BMSC’s) and fibronectin coated plates. Migration to 30nM SDF1-α was assessed using transwell plates comparing ST3GAL6 knockdown cells to scrambled controls. The commercially available sialyltransferase inhibitor 3Fax-Neu5Ac was used to pre-treat MM cells in vitro prior to assessment of apoptosis by flow cytometry. shST3GAL6 MM1s cells positive for green fluorescent protein and luciferin (GFP-Luc+) were injected into tail veins of SCID-Bg mice (5x106 cells, n=5/group) and mice were followed weekly using bioluminescent imaging (BLI) for tumor development. Bone marrow homing of tumor cells was assessed using in vivoconfocal imaging of the skull vasculature (n=3/group). Results Knockdown of ST3GAL6 in MM cell lines resulted in a 50% reduction in cell surface staining with the monoclonal antibody HECA-452. This indicated reduced expression of cutaneous lymphocyte associated antigen (CLA), a carbohydrate domain shared by sialyl Lewis X (sLex) and sialyl Lewis a (sLea) antigens, confirming suppression of ST3GAL6 activity. There was a significant reduction in the ability of knockdown cells to adhere to BMSC’s and fibronectin in-vitro compared to scrambled controls (P=0.016, 0.032 respectively). Migration ability of these cells in response to SDF1-α was also reduced (P=0.01). In vivo in a xenograft SCID-Bg mouse model shST3GAL6 cells demonstrated a reduced tumor burden as assessed by weekly BLI (P=0.017 at week 4). A consolidated map of the skull bone marrow niche in mice injected with shST3GAL6 MM1s GFP-Luc+ cells revealed a reduced homing ability of these cells in comparison to mice injected with scrambled control cells. Treatment of the MM cell lines MM1s and RPMI-8226 with a sialyltransferase inhibitor 3Fax-Neu5Ac resulted in almost complete elimination of cell surface sLex and/or sLea expression as determined by HECA-452 staining. Following pre-treatment with 3Fax-Neu5Ac, MM1S cells grown in co-culture with BMSC’s cells showed increased sensitivity to Bortezomib compared to cells treated with bortezomib alone. Conclusions shRNA knockdown of ST3GAL6 in MM cells significantly inhibits adhesion and migration in vitro with reduced homing and proliferation potential in vivo. In conjunction with the results of enzymatic inhibition this indicates that sialylation may play an important role in the malignant behavior of MM cells. Studies are ongoing to address the potential role of altered glycosylation in MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

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